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The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment
Staggs, S., E. Beckman, S. Keely, R. Mackwan, M. Ware, A. Moyer, J. Ferretti, A. Sayed, L. Xiao, AND E. Villegas. The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment. PLoS ONE . Public Library of Science, San Francisco, CA, 8(6):e66562, (2013).
Purpose of this study was to determine the use of molecular-based detection assays to quantitate Cryptosporidium oocysts for regulatory purposes, i.e. to replace microscopic enumeration as described in Method 1623
Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as for species identification. Both assays have been used to differentiate between Cryptosporidium species, particularly those that are infectious to humans. The purpose of this study was to evaluate sensitivity and specificity of TaqMan-based quantitative real-time PCR assays for detecting Cryptosporidium spp., Cryptosporidium parvum, or Cryptosporidium hominis oocysts in water. A total of ten assays from previously published studies or newly developed in-house assays were analyzed for specificity, cross-reactivity, and sensitivity. Results revealed that all ten assays have varying degrees of specificity. A single flow-sorted oocyst in reagent water can be detected; however, sensitivity decreased to 10 oocysts in the presence of an environmental matrix. Interestingly, select qPCR assays designed to detect all Cryptosporidium spp. using the 18S rRNA gene cross-amplified fungi, green algae, and dinoflagellate species. More importantly, qPCR assays did not have the resolution to reliably differentiate between 1, 2, or 5 oocysts in a sample. Overall, this study revealed that qPCR-based assays can be useful for detecting the presence of human infectious Cryptosporidium species; however unequivocally determining low levels of oocysts typically found in the environment may prove more difficult.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL EXPOSURE RESEARCH LABORATORY
MICROBIOLOGICAL AND CHEMICAL EXPOSURE ASSESSMENT DIVISION
BIOHAZARD ASSESSMENT RESEARCH BRANCH